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Ictalurus Punctatus Activation-Induced Cytidine Deaminase Expression
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- Author / Creator
- Saunders, Holly L.
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Antigen-specific recognition and memory, the hallmarks of conventional mammalian adaptive immunity, are found in all jawed vertebrates along with all elements central to cognate adaptive immune responses (e.g. B-cells). During T-cell-dependent humoral immune responses in mammals, antigen-specific recognition is significantly enhanced by antibody affinity maturation. The mammalian antibody affinity maturation paradigm, derived from mouse/human, involves genetic modification of the immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) coupled with phenotypic selection for B-cells that express higher-affinity antibody. Antigen-activated B-cells undergo these processes while sequestered in germinal centers, which are transient, specialized microenvironments that develop in secondary lymphoid tissues. These histologically obvious sites also harbour cognate T-cells and follicular dendritic cells in an organization that is believed to be essential to efficient selection and, consequently, to substantial increases in antibody affinity.
Although SHM and CSR are discrete events, they share several features including an indispensable requirement for the mutator enzyme, activation-induced cytidine deaminase (AID). In germinal center B-cells, the AID enzyme deaminates cytosine to uracil in single-stranded (ssDNA) of the transcribed Ig genes. This pre-mutagenic uracil DNA lesion is a target of ubiquitously expressed DNA damage tolerance and repair molecules, which, ultimately, achieve SHM and CSR outcomes.
Information about the immune response in lower vertebrates (sharks, bony fishes, amphibians) is relatively sparse, but they appear to exhibit limited if any antibody affinity maturation. Although conventional germinal centers have not been detected in lower vertebrates, SHM has been detected in sharks and both SHM and CSR have been detected in amphibians. In the teleost bony fishes, CSR cannot occur due to the absence of additional constant regions in the Ig heavy chain (IgH) gene, but their SHM status is unknown.
To better understand antibody affinity maturation in teleosts, the following questions need to be answered: Are teleosts able to somatically hypermutate their Ig genes? And, if so, how do teleosts balance the risks and benefits (i.e. prevent autoimmunity versus improve antibody affinity) of this highly mutagenic process? To address these questions, we sought an AID homologue in the channel catfish (Ictalurus punctatus), a representative teleost. Since AID is the only B-cell-specific factor required for SHM in mammals, the identification of an AID homologue in catfish could be used to determine if teleosts are capable of SHM. It could also be used as a marker to specifically identify in situ the teleost equivalents of mammalian germinal center B-cells as indicators of sites of possible germinal center analogues.
We found two lines of evidence that support the proposal that catfish undergo AID-mediated SHM. First, catfish express an AID homologue in a tissue expression pattern that is consistent with AID-mediated mutation playing a role in immune function. The catfish AID homologue has 57% amino acid identity and 81% amino acid similarity with mouse/human AIDs and maintains strong conservation of amino acids that have been determined to be important for cytidine deaminase, SHM, and CSR functions. After native AID gene expression had been upregulated in a catfish immortal B-cell line, DNA sequence comparison of the endogenous IgH gene variable region exon in subclones revealed the presence of SHM with the mutation signature of AID. Thus, catfish AID is catalytically active in vivo and, as in mammals, is targeted to Ig genes.
Second, cell aggregates observed in catfish spleen sections appear to be broadly reminiscent of mammalian germinal centers. Distinct cells that express AID mRNA colocalize in clusters with melanomacrophage cells, which trap antigen and are putative teleost analogues of mammalian follicular dendritic cells. That B-cells undergo AID-mediated SHM at sites where antigen is “trapped” suggests the presence of concomitant phenotypic selection. However, the intense selection that occurs in mammalian germinal centers is accompanied by crucial features, such as extensive B-cell apoptosis, that appear to be absent in catfish. Notably, in mouse, blocking B-cell apoptosis in germinal centers severely diminishes antibody affinity maturation.
In conclusion, we have shown that catfish diversify their Ig genes via AID-dependent SHM and that cells that express AID reside in distinct cellular aggregations partially reminiscent of mammalian germinal centers. Since catfish can somatically modify their Ig genes, the apparently lesser antibody affinity maturation of teleosts may result from relatively inefficient selection for higher-affinity variants. -
- Graduation date
- Fall 2020
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.